Compositions and methods for treating neoplastic disease using chemotherapy and radiation sensitizers

ABSTRACT

Inhibitors of KIAA0175 are provided that reduce the expression or biological activities of KIAA0175, p53 and/or p21 in a mammalian cell. KIAA0175 inhibitors include anti-sense molecules, ribozymes, antibodies and antibody fragments, proteins and polypeptides as well as small molecules. KIAA0175 inhibitors find use in compositions and methods for decreasing KIAA0175, p53 and/or p21 gene expression as well as methods for increasing the chemo and/or radiosensitivity of mammalian cells, including tumor cells, methods for decreasing the side effects of cancer therapy and methods for treating neoplastic diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.09/870,937 filed May 30, 2001, which claims priority from U.S. PatentApplication No. 60/208,435 filed May 31, 2000, which is incorporated byreference herein in its entirety.

TECHNICAL FIELD

This invention relates generally to regulation of the cell cycle andcell death following exposure to agents that cause DNA damage. Morespecifically, the present invention provides compositions and methodsfor inhibiting KIAA0175 gene expression and/or biological activity aswell as for reducing the expression and/or activation of p53, p21 orrelated proteins. Such compositions and methods are useful aschemotherapy and radiation sensitizers and, as a consequence, findutility in the treatment of neoplastic disease.

BACKGROUND OF THE INVENTION

Traditional chemo and radiotherapy for the treatment of tumors islimited in utility by the absence of target specificity. Methodologiesemploying targeted radiotherapy wherein radioisotopes are conjugated totumor-seeking agents have been attempted to improve biologicalspecificity. Unfortunately, very few selective agents are available forthe many tumor types and where selective agents do exist, thetherapeutic advantage has been minimal. Recent methodologies fortreating neoplastic diseases attempt to improve specificity by employinga class of molecules called “sensitizers” that increase the sensitivityof treated cells to, for example, chemotherapy and γ-irradiation. Forexample, administration of drugs such as 5-fluorouracil and wortmanninhas been attempted in order to increase cellular radiation sensitivity.Owing to the non-specific nature of these and other currently available“sensitizers,” however, normal, non-neoplastic cells are subject toincreased chemotherapy and radiation sensitivity thus resulting in ahigh level of cellular toxicity. What is urgently needed arechemotherapy and radiation sensitizers having improved specificityallowing preferential sensitivity of neoplastic as compared to normalcells. The KIAA0175 inhibitors of the present invention fulfill theseand other related needs.

SUMMARY OF THE INVENTION

The present invention provides, in one embodiment, inhibitors ofKIAA0175. Inventive inhibitors include, but are not limited to,anti-sense molecules, ribozymes, antibodies or antibody fragments,proteins or polypeptides as well as small molecules. Exemplaryanti-sense molecules comprise at least 16, 17, 20 or 25 consecutivenucleotides of or hybridize under stringent conditions to the nucleicacid of SEQ ID NO:9. More preferred are anti-sense molecules thatcomprise at least 25 consecutive nucleotides of or hybridize understringent conditions to the sequence of SEQ ID NO:9. Representativeanti-sense molecules are provided herein as SEQ ID NO:1, SEQ ID NO:3,and SEQ ID NO:5.

In further embodiments, compositions are provided that comprise one ormore KIAA0175 inhibitor in a pharmaceutically acceptable carrier.

Additional embodiments provide methods of decreasing KIAA0175 geneexpression or biological activity.

Still further embodiments provide methods for decreasing the expressionof p53 and methods for decreasing the expression of p21.

Other embodiments provide methods of increasing the chemo and/orradiosensitivity of a mammalian cell, methods of reducing the sideeffects of cancer therapy and other types of stress associated with p53induction and methods of treating neoplastic disease.

Each of the methods of the present invention have in common theadministration of one or more inventive KIAA0175 inhibitor to amammalian cell.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram of various KIAA0175 two-hybrid bait constructs inthe LexA plasmid vector pBTM 116 and in the Gal4BD plasmid vectorpAS2-1.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention is directed generally toregulation of the cell cycle and cell death following exposure to agentsthat cause DNA damage. More specifically, the invention disclosed hereinprovides inhibitors of KIAA0175, including anti-sense polynucleotidesand ribozymes, proteins or polypeptides, antibodies or fragments thereofand small molecules; compositions comprising KIAA0175 inhibitors;methods of reducing p53 and/or p21 activation and/or or gene expression;methods of increasing the chemo and/or radiation sensitivity of amammalian cell, methods of reducing the side effects of cancer therapyas well as methods of treating neoplastic disease. Each of these methodshave in common the administration to a mammalian cell of one or moreKIAA0175 inhibitor.

The p53 tumor suppressor is the most commonly mutated gene in humancancer. In normal cells under physiological conditions, p53 is expressedat low levels and has a short half-life. When cells are challenged byany of a variety of stress inducing stimuli, such as DNA damage,hypoxia, nucleotide deprivation, viral infection, oncogene activation,or factors that induce cell cycle arrest and cell death, p53 becomesstabilized and activated. Under such conditions, p53 acts as the“guardian of the genome” by facilitating genomic stability which isaccomplished through the stalling of cellular growth and division at avariety of cell cycle checkpoints.

Eukaryotes from yeast to man have developed surveillance mechanisms thatrespond to DNA damage. In mammals, this surveillance mechanism includesa checkpoint, involving the ataxia telangiectasia mutated (ATM), p53 andp21 genes, that arrests cells in the G1 phase. For example, when cellsare exposed to γ-irradiation, p53 becomes an active transcription factorthat participates in the expression of various genes, including p21,that are important in regulating the cell cycle at the G1 to S phasecheckpoint. El-Deiry, W. S. Cancer Biology 8:345-357 (1998); Cox, L. S.et al., Bioessays 17:501 (1995); Sherr, C. J., Science 274:1672 (1996).

Ataxia Telangiectasia (A-T) is an autosomal recessive disordercharacterized by progressive cerebellar ataxia, neuronal degeneration,hypersensitivity to ionizing radiation (IR) and a high risk of cancer.Individuals afflicted with A-T exhibit acute sensitivity to ionizingradiation and radiomimetic chemicals, and their cell cycle checkpointsfail to be activated after treatment with these agents. Sadvitsky, K. etal., Science 268:1749 (1995). Thus, for example, when cell survival isused as an end-point, A-T fibroblasts and lymphoblasts are 3-4-fold moresensitive to ionizing radiation as compared to wild-type controls. Chen,P. C. et al., Nature 274:484-486 (1978) and Lehman, A. R. et al., In.“Ataxia-Telangiectasia—A Cellular and Molecular Link Between Cancer,Neurophathology and Immune Deficiency,” pp. 347-353. (Ed. Bridges, B. A.and Harnden, D. G., Wiley, N.Y., 1982). Similarly, A-T cells are alsohypersensitive to radiomimetic agents such as neocarcinostatin andbleomycin.

A-T is caused by a defect in the ATM gene the product of which has beenimplicated in both post-translational activation and increasedexpression of p53. Banin, et al., Science 281:1674-1677 (1998); Canman,et. al., Science 281:1677-1679 (1998). ATM has a phosphoinositide3-kinase-related domain and a wortmannin-sensitive protein kinaseactivity, Keith, C. T. et al., Science 270:50 (1995), and phosphorylatesp53 on a single amino acid residue, serine-15, in response toγ-irradiation. Cells from patients afflicted with A-T, resulting from anabsence of ATM, show a reduced and delayed activation of p53 in responseto DNA damage as evidenced by diminished p53 phosphorylation. Kastan, M.B. et al., Cell 71:587 (1992); Lu, X. et al., Cell 75:765 (1993);Siliciano, J. D. et al., Genes Dev. 11:3471 (1997) and Shieh, S.-Y. etal., Cell 91:325 (1997). Mutation of p53 at serine-15 reduces theability of p53 to arrest cell growth. Fiscella, M. et al., Oncogene8:1519 (1993).

In its normal, unphosphorylated state, p53 associates with the MDM2protein which acts as a negative regulator of p53 stability by targetingit for proteolysis. Kubbutat, H. H. G. et al., Nature 387:299-303(1997); Haupt, Y. et al., Nature 387:296-299 (1997); and Kubbutat, M. H.G. et al., Mol. Cell. Biol. 18:5690-5698 (1998). MDM2 binding alsoconceals p53's transactivation domain thereby blocking p53-dependenteffects in cell cycle inhibition and apoptosis. Under conditions ofcellular stress, such as challenge with γ-irradiation, phosphorylationat serine-1 S reduces MDM2's p53 binding affinity preventing MDM2mediated inhibition of p53-specific transcription as well asproteosome-mediated p53 degradation. It is for this reason that it hasbeen suggested that p53 phosphorylation at serine-15 is important forgenomic stability. Momand, J. et al., Cell 69:1237 (1992); Oliner, J.D., et al., Nature 362:857 (1993); Haupt, Y. et al., Nature 387:286(1997); and Kubbutat, M. H. G. et al., Nature 387:299 (1997).

Arrest of the cell cycle at the G1 stage occurs, in part, throughtranscriptional activation of p21, a tight-binding inhibitor of Cdksthat control entry into the S phase. Elledge, J. et al., Trends CellBiol. 6:388 (1996). It has been shown that the absence of p21 activityincreases cellular sensitivity to ionizing radiation and delays theonset of lymphoma in ATM-deficient mice. Wang, Y. A. et al., Proc. Natl.Acad. Sci. U.S.A. 94:14590-14595 (1997).

Checkpoint signal transduction was originally described in the yeastSaccharomyces cerevisiae. Weinert, T. A., et al., Science 241:317(1988). Two genes, MEC1 and RAD53, are essential in the control of theyeast checkpoint. Mec1 is a member of the phosphatidyl inositol kinasesuperfamily and is believed to function as a protein kinase. Rad53 is aprotein kinase that is phosphorylated and activated in response to DNAdamage. Phosphorylation of Rad53 is dependent upon, inter alia, Mec1indicating that Rad53 functions downstream of Mec1 to transduce thesignal from DNA damage.

The true mammalian homologue to Saccharomyces cerevisiae RadS3 has beenidentified as the cell cycle regulatory protein Chk2 kinase. Matsuoka,S. et al., Science 282:1893-1897 (1998). As part of the presentinvention it was found that Rad53 is also homologous to a cDNA sequencefirst identified by Nagase, T. et al. and referred to as KIAA0175. DNAResearch 3:17-24 (1996). These authors observed that KIAA0175 contains aregion of homology with the family of serine-threonine protein kinasesin particular the Xenopus laevis p69Eg3 protein kinase.

As disclosed herein, the KIAA0175 gene is expressed in a variety oftissues including testis, thymus, colon, placenta and spleen. KIAA0175is also expressed in a number of cancer cell lines such as MOLT-4, HeLa,S3, K562, SW480, HL-60 and Raji cells. See Example 1 and FIGS. 1A-C.

The present invention also discloses that the protein product of theKIAA0175 gene possesses an autophosphorylation activity which activitydepends upon, inter alia, a lysine residue at amino acid position 40.Thus, e.g., Cos7 cells transfected with a plasmid construct comprisingan HA-tagged KIAA0175 wild-type or lysine to alanine substitution atposition 40 (i.e., K40A) express the KIAA0175 protein but only thewild-type protein retains autophosphorylation activity as determined bythe incorporation of ³²P when immunoprecipitated protein is incubated inthe presence of γ-³²P-ATP. See Example 2, FIGS. 2A-B. It is alsoobserved that, following exposure of cells to either γ-irradiation orhydroxyurea, the levels of both endogenously expressed and recombinantKIAA0175 protein increase. FIGS. 3 and 4.

Radiation Sensitizers

Cells defective in ATM as well as in other members of thephosphatidylinositol-3-kinase (PI3K) family involved in regulating DNAdamage dependent cell cycle checkpoints, such as ATR and DNA-PK, exhibithypersensitivity to ionizing radiation and other DNA-damaging agents. Ithas been suggested that inhibitors of one or more of these kinases mightenhance the cytotoxic effects of ionizing radiation or DNA-damagingcancer chemotherapeutic drugs by sensitizing cells to these agents.Sarkaria, J. N. et al., Cancer Res. 58:4375-4382 (1998) and Hosoi, Y. etal., Int. J. Cancer 78:642-647 (1998). This suggestion is supported bythe observation that the sterol-like fungal metabolite, wortmannin, isan effective radiosensitizer. Chemikova, S. B. et al., Rad. Res.151:159-166 (1999); Boulton, S. et al., Carcinogenesis (Lond.)17:2285-2290 (1996); Price, B. D. et al., Cancer Res. 56:246-250 (1996);and Rosenzweig, K. E. et al., Clin. Cancer Res. 3:1149-1156 (1997).Wortmannin irreversibly inhibits the lipid kinase activities ofmammalian PI-3Ks by covalent modification of a critical lysine residuein their phosphotransferase domains. Powis, G. et al., Cancer Res.54:2419-2423 (1994) and Wymann, M. P. et al., Mol. Cell. Biol.16:1722-1733 (1996).

Traditional radiosensitizers such as 5-fluorouracil either enhance thelevel of initial DNA damage caused by radiation or impede the repair ofradiation-induced DNA lesions by inhibiting enzymes involved in DNAmetabolism, synthesis and repair. Sarkaria, et al., supra. The intrinsiccytotoxicity associated with the administration of such drugs limitstheir clinical utility as radiation and chemotherapeutic agentsensitizers. Similarly, because wortmannin acts non-specifically byinhibiting a broad range of PI3K related proteins, the risks associatedwith traditional radiosensitizers and chemosensitizers may also apply tothe use of wortmannin. What is needed in the art are highly specificinhibitors of a DNA damage signaling pathway associated protein that isinvolved in cell cycle regulation. The present invention fulfills thisneed by providing specific inhibitors of KIAA0175 gene expression and/orbiological activity. Such inhibitors abrogate cell cycle arrest andabolish the necessary damage repair, thus leaving cells vulnerable tothe cytotoxicity of DNA damaging agents such as, e.g., ionizingradiation.

Inhibitors of KIAA0175 are Effective in Reducing KIAA0175, p53 and p21Gene Expression

The present invention provides inhibitors of KIAA0175. Inventiveinhibitors include anti-sense molecules and ribozymes, proteins orpolypeptides, antibodies or fragments thereof as well as smallmolecules. Each of these KIAA0175 inhibitors share the common featurethat they reduce the expression and/or biological activity of KIAA0175and, as a consequence, diminish the expression and/or activation of p53and/or p21. In addition to the exemplary KIAA0175 inhibitors disclosedherein, alternative inhibitors may be obtained through routineexperimentation utilizing methodology either specifically disclosedherein or as otherwise readily available to and within the expertise ofthe skilled artisan.

Anti-Sense Molecules and Ribozymes

As discussed above, KIAA0175 inhibitors of the present invention includeanti-sense molecules that, when administered to mammalian cells, areeffective in reducing, for example, intracellular protein levels of p53and/or p21. Anti-sense molecules bind in a sequence-specific manner tonucleic acids, such as mRNA or DNA. When bound to mRNA that hascomplementary sequences, anti-sense molecules prevent translation of themRNA (see, e.g., U.S. Pat. No. 5,168,053 to Altman et al.; U.S. Pat. No.5,190,931 to Inouye, U.S. Pat. No. 5,135,917 to Burch; U.S. Pat. No.5,087,617 to Smith and Clusel et al. Nucl. Acids Res. 21:3405-3411(1993), which describes dumbbell anti-sense oligonucleotides).

Anti-sense technology can be used to control gene expression throughtriple-helix formation, which promotes the ability of the double helixto open sufficiently for the binding of polymerases, transcriptionfactors or regulatory molecules. See Gee et al., In Huber and Carr,“Molecular and Immunologic Approaches,” Futura Publishing Co. (Mt.Kisco, N.Y.; 1994). Alternatively, an anti-sense molecule may bedesigned to hybridize with a control region of the KIAA0175 gene, e.g.,promoter, enhancer or transcription initiation site, and blocktranscription of the gene; or block translation by inhibiting binding ofa transcript to ribosomes. See generally, Hirashima et al. in MolecularBiology of RNA. New Perspectives (M. Inouye and B. S. Dudock, eds., 1987Academic Press, San Diego, p. 401); Oligonucleotides. Anti-senseInhibitors of Gene Expression (J. S. Cohen, ed., 1989 MacMillan Press,London); Stein and Cheng, Science 261:1004-1012 (1993); WO 95/10607;U.S. Pat. No. 5,359,051; WO 92/06693; and EP-A2-612844 each of which isincorporated herein by reference.

Briefly, such molecules are constructed such that they are complementaryto, and able to form Watson-Crick base pairs with, a region oftranscribed KIAA0175 mRNA sequence. The resultant double-strandednucleic acid interferes with subsequent processing of the mRNA, therebypreventing protein synthesis.

In general, a portion of a sequence complementary to the KIAA0175 codingregion may be used to modulate gene expression. The nucleic acidsequence of the human KIAA0175 cDNA is described in Nagase, T. et al.,DNA Research 3:17-24 (1996), incorporated herein by reference, and ispresented herein as SEQ ID NO:9. Alternatively, cDNA constructs that canbe transcribed into anti-sense RNA may be introduced into cells ortissues to facilitate the production of anti-sense RNA. Thus, as usedherein, the phrase “anti-sense molecules” broadly encompasses anti-senseoligonucleotides whether synthesized as DNA or RNA molecules as well asall plasmid constructs that, when introduced into a mammalian cell,promote the production of anti-sense RNA molecules. An anti-sensemolecule may be used, as described herein, to inhibit expression ofKIAA0175, p53 or p21 genes as well as any other gene that requiresKIAA0175 for its expression.

Anti-sense molecules for use as described herein can be synthesized byany method known to those of skill in this art including chemicalsynthesis by, for example, solid phase phosphoramidite chemicalsynthesis. See, e.g., WO 93/01286; U.S. Pat. No. 6,043,090; U.S. Pat.No. 5,218,088; U.S. Pat. No. 5,175,269; and U.S. Pat. No. 5,109,124,each of which is incorporated herein by reference. Alternatively, RNAmolecules may be generated by in vitro or in vivo transcription of DNAsequences encoding the KIAA0175 cDNA, or a portion thereof, providedthat the DNA is incorporated into a vector downstream of a suitable RNApolymerase promoter (such as, e.g., T3, T7 or SP6). Large amounts ofanti-sense RNA may be produced by incubating labeled nucleotides with alinearized KIAA0175 cDNA fragment downstream of such a promoter in thepresence of the appropriate RNA polymerase. Such anti-sense moleculesare preferably at least 16, 18 or 20 nucleotides in length as well asall intermediate lengths there between. More preferably, anti-sensemolecules are at least 25 nucleotides in length. A further embodiment ofthe present invention provides anti-sense molecules that are at least30, 40, 50, or 75 nucleotides in length, as well as all intermediatelengths there between. Within certain embodiments, an anti-sensemolecule of the present invention will comprise a sequence that isunique to the KIAA0175 cDNA sequence of SEQ ID NO:9 or that canhybridize to the cDNA of SEQ ID NO:9 under conditions of highstringency. Within the context of the present invention, high stringencymeans standard hybridization conditions such as, e.g., 5×SSPE, 0.5% SDSat 65° C. or the equivalent thereof. See Sambrook et al., supra andMolecular Biotechnology: Principles and Applications of Recombinant DNA,supra incorporated herein by reference.

Anti-sense oligonucleotides are typically designed to resist degradationby endogenous nucleolytic enzymes by using such linkages as:phosphorothioate, methylphosphonate, sulfone, sulfate, ketyl,phosphorodithioate, phosphoramidate, phosphate esters, and other suchlinkages (see, e.g., Agrwal et al., Tetrehedron Lett. 28:3539-3542(1987); Miller et al., J. Am. Chem. Soc. 93:6657-6665 (1971); Stec etal., Tetrehedron Lett. 26:2191-2194 (1985); Moody et al., Nuc. AcidsRes. 12:4769-4782 (1989); Uznanski et al., Nucl. Acids Res.17(12):4863-4871 (1989); Letsinger et al., Tetrahedron 40:137-143(1984); Eckstein, Annu. Rev. Biochem. 54:367-402 (1985); Eckstein,Trends Biol. Sci. 14:97-100 (1989); Stein, in: Oligodeoxynucleotides.Anti-sense Inhibitors of Gene Expression, Cohen, Ed, Macmillan Press,London, pp. 97-117 (1989); Jager et al., Biochemistry 27:7237-7246(1988)). Possible additional or alternative modifications include, butare not limited to, the addition of flanking sequences at the 5′ and/or3′ ends and/or the inclusion of nontraditional bases such as inosine,queosine and wybutosine, as well as acetyl- methyl-, thio- and othermodified forms of adenine, cytidine, guanine, thymine and uridine.

Within alternate embodiments of the present invention, KIAA0175inhibitors may be ribozymes. A ribozyme is an RNA molecule thatspecifically cleaves RNA substrates, such as mRNA, resulting in specificinhibition or interference with cellular gene expression. As usedherein, the term “ribozymes” includes RNA molecules that containanti-sense sequences for specific recognition, and an RNA-cleavingenzymatic activity. The catalytic strand cleaves a specific site in atarget RNA at greater than stoichiometric concentration.

A wide variety of ribozymes may be utilized within the context of thepresent invention, including for example, the hammerhead ribozyme (forexample, as described by Forster and Symons, Cell 48:211-220 (1987);Haseloff and Gerlach, Nature 328:596-600 (1988); Walbot and Bruening,Nature 334:196 (1988); Haseloff and Gerlach, Nature 334:585 (1988)); thehairpin ribozyme (for example, as described by Haseloff et al., U.S.Pat. No. 5,254,678, issued Oct. 19, 1993 and Hempel et al., EuropeanPatent Publication No. 0 360 257, published Mar. 26, 1990); andTetrahymena ribosomal RNA-based ribozymes (see Cech et al., U.S. Pat.No. 4,987,071). Ribozymes of the present invention typically consist ofRNA, but may also be composed of DNA, nucleic acid analogs (e.g.,phosphorothioates), or chimerics thereof (e.g., DNA/RNA/RNA).

Ribozymes can be targeted to any RNA transcript and can catalyticallycleave such transcripts (see, e.g., U.S. Pat. No. 5,272,262; U.S. Pat.No. 5,144,019; and U.S. Pat. Nos. 5,168,053, 5,180,818, 5,116,742 and5,093,246 to Cech et al.). According to certain embodiments of theinvention, any such KIAA0175 mRNA-specific ribozyme, or a nucleic acidencoding such a ribozyme, may be delivered to a host cell to effectinhibition of KIAA0175 gene expression. Ribozymes and the like maytherefore be delivered to the host cells by DNA encoding the ribozymelinked to a eukaryotic promoter, such as a eukaryotic viral promoter,such that upon introduction into the nucleus, the ribozyme will bedirectly transcribed.

Proteins and Polypeptides

In addition to the anti-sense molecules and ribozymes disclosed herein,supra, KIAA0175 inhibitors of the present invention also includeproteins or polypeptides that are effective in either reducing KIAA0175gene expression or in decreasing one or more of KIAA0175's biologicalactivities. A variety of methods are readily available in the art bywhich the skilled artisan may, through routine experimentation, rapidlyidentify such KIAA0175 inhibitors. The present invention is not limitedby the following exemplary methodologies.

As discussed above, KIAA0175 is an active protein kinase that possessesan autophosphorylation activity. Thus, inhibitors of KIAA0175'sbiological activities encompass those proteins and/or polypeptides thatinterfere with KIAA0175's kinase activity. Such interference may occurthrough direct interaction with KIAA0175's kinase domain or indirectlythrough non- or un-competitive inhibition such as via binding to anallosteric site. Accordingly, available methods for identifying proteinsand/or polypeptides that bind to KIAA0175 may be employed to identifylead compounds that may, through the methodology disclosed herein, seeinfra, be characterized for their KIAA0175 inhibitory activity and/orefficacy as radiation sensitizers.

A vast body of literature is available to the skilled artisan thatdescribes methods for detecting and analyzing protein-proteininteractions. Reviewed in Phizicky, E. M. et al., MicrobiologicalReviews 59:94-123 (1995) incorporated herein by reference. Such methodsinclude, but are not limited to physical methods such as, e.g., proteinaffinity chromatography, affinity blotting, immunoprecipitation andcross-linking as well as library-based methods such as, e.g., proteinprobing, phage display and two-hybrid screening. Other methods that maybe employed to identify protein-protein interactions include geneticmethods such as use of extragenic suppressors, synthetic lethal effectsand unlinked noncomplementation. Exemplary methods are described infurther detail below.

Inventive KIAA0175 inhibitors may be identified through biologicalscreening assays that rely on the direct interaction between theKIAA0175 protein and a panel or library of potential inhibitor proteins.Biological screening methodologies, including the various “n-hybridtechnologies,” are described in, for example, Vidal, M. et al., Nucl.Acids Res. 27(4):919-929 (1999); Frederickson, R. M., Curr. Opin.Biotechnol. 9(1):90-6 (1998); Brachmann, R. K. et al., Curr. Opin.Biotechnol. 8(5):561-568 (1997); and White, M. A., Proc. Natl. Acad.Sci. U.S.A. 93:10001-10003 (1996) each of which is incorporated hereinby reference.

The two-hybrid screening methodology may be employed to search new orexisting target cDNA libraries for KIAA0175 binding proteins that haveinhibitory properties. The two-hybrid system is a genetic method thatdetects protein-protein interactions by virtue of increases intranscription of reporter genes. The system relies on the fact thatsite-specific transcriptional activators have a DNA-binding domain and atranscriptional activation domain. The DNA-binding domain targets theactivation domain to the specific genes to be expressed. Because of themodular nature of transcriptional activators, the DNA-binding domain maybe severed covalently from the transcriptional activation domain withoutloss of activity of either domain. Furthermore, these two domains may bebrought into juxtaposition by protein-protein contacts between twoproteins unrelated to the transcriptional machinery. Thus, two hybridsare constructed to create a functional system. The first hybrid, i.e.,the bait, consists of a transcriptional activator DNA-binding domainfused to a protein of interest. The second hybrid, the target, iscreated by the fusion of a transcriptional activation domain with alibrary of proteins or polypeptides. Interaction between the baitprotein and a member of the target library results in the juxtapositionof the DNA-binding domain and the transcriptional activation domain andthe consequent up-regulation of reporter gene expression.

A variety of two-hybrid based systems are available to the skilledartisan that most commonly employ either the yeast Gal4 or E. coli LexADNA-binding domain (BD) and the yeast Gal4 or herpes simplex virus VP16transcriptional activation domain. Chien, C.-T. et al., Proc. Natl.Acad. Sci. U.S.A. 88:9578-9582 (1991); Dalton, S. et al., Cell68:597-612 (1992); Durfee, T. K. et al., Genes Dev. 7:555-569 (1993);Vojtek, A. B. et al., Cell 74:205-214 (1993); and Zervos, A. S. et al.,Cell 72:223-232 (1993). Commonly used reporter genes include the E. colilacZ gene as well as selectable yeast genes such as HIS3 and LEU2.Fields, S. et al., Nature (London) 340:245-246 (1989); Durfee, T. K.,supra; and Zervos, A. S., supra. A wide variety of activation domainlibraries are readily available in the art such that the screening forinteracting proteins may be performed through routine experimentation.

Suitable bait proteins for the identification of KIAA0175 interactingproteins may be designed based on the KIAA0175 cDNA sequence presentedherein as SEQ ID NO:9. Such bait proteins include either the full-lengthKIAA0175 protein or fragments thereof. For example, for screening ofinhibitors that block KIAA0175's autophosphorylation activity, it may beadvantageous to construct bait proteins that include lysine-40 and thatencompass flanking amino acids thereto that comprise the protein'skinase domain. Exemplary KIAA0175 bait proteins, expressed as eitherLexA or Gal4BD, are depicted in FIG. 10. Representative bait constructsinclude the coding region for KIAA0175's kinase domain (e.g., the LexAconstructs 2, 8 and 61 as well as the Gal4BD constructs 24, 29 and 37).In addition, LexA constructs 44 and 54 and Gal4BD constructs 27 and 33each bear the single amino acid substitution within the kinase domain atK40A. FIG. 10 also depicts alternative bait constructs encoding KIAA0175polypeptide fragments outside the kinase domain (e.g., the Lex Aconstructs 14 and 19 as well as the Gal4BD constructs 36 and 42).

Plasmid vectors, such as, e.g., pBTM116 and pAS2-1, for preparingKIAA0175 bait constructs and target libraries are readily available tothe artisan and may be obtained from such commercial sources as, e.g.,Clontech (Palo Alto, Calif.), Invitrogen (Carlsbad, Calif.) andStratagene (La Jolla, Calif.). These plasmid vectors permit the in-framefusion of cDNAs with the DNA-binding domains as LexA or Gal4BD,respectively.

KIAA0175 inhibitors of the present invention may alternatively beidentified through one of the physical or biochemical methods availablein the art for detecting protein-protein interactions.

Through the protein affinity chromatography methodology, lead compoundsto be tested as potential KIAA0175 inhibitors may be identified byvirtue of their specific retention to KIAA0175 when either covalently ornon-covalently coupled to a solid matrix such as, e.g., Sepharose beads.The preparation of protein affinity columns is described in, forexample, Beeckmans, S. et al., Eur. J. Biochem. 117:527-535 (1981) andFormosa, T. et al., Methods Enzymol. 208:24-45 (1991). Cell lysatescontaining the full complement of cellular proteins may be passedthrough the KIAA0175 affinity column. Proteins having a high affinityfor KIAA0175 will be specifically retained under low-salt conditionswhile the majority of cellular proteins will pass through the column.Such high affinity proteins may be eluted from the immobilized KIAA0175under conditions of high-salt, with chaotropic solvents or with sodiumdodecyl sulfate (SDS). In some embodiments, it may be preferred toradiolabel the cells prior to preparing the lysate as an aid inidentifying the KIAA0175 specific binding proteins. Methods forradiolabeling mammalian cells are well known in the art and areprovided, e.g., in Sopta, M. et al., J. Biol. Chem. 260:10353-10360(1985).

Suitable KIAA0175 proteins for affinity chromatography may be fused to aprotein or polypeptide to permit rapid purification on an appropriateaffinity resin. For example, the KIAA0175 cDNA may be fused to thecoding region for glutathione S-transferase (GST) which facilitates theadsorption of fusion proteins to glutathione-agarose columns. Smith etal., Gene 67:31-40 (1988). Alternatively, fusion proteins may includeprotein A, which can be purified on columns bearing immunoglobulin G;oligohistidine-containing peptides, which can be purified on columnsbearing Ni²⁺; the maltose-binding protein, which can be purified onresins containing amylose; and dihydrofolate reductase, which can bepurified on methotrexate columns. One exemplary tag suitable for thepreparation of KIAA0175 fusion proteins that is presented herein is theepitope for the influenza virus hemagglutinin (HA) against whichmonoclonal antibodies are readily available and from which antibodies anaffinity column may be prepared.

In those cases where candidate KIAA0175 inhibitors are directed againstKIAA0175's kinase domain, it may be advantageous to phosphorylate theKIAA0175 protein prior to preparing the affinity column. As disclosed inExample 2, KIAA0175 may be phosphorylated when immobilized on a solidsurface. Suitable phosphorylation conditions include 10 μM ATP, 1 mMDTT, 10 mM MgCl₂, 10 mM MnCl₂ and 50 mM Tris, pH 7.5.

Proteins that are specifically retained on a KIAA0175 affinity columnmay be identified after subjecting to SDS polyacrylamide gelelectrophoresis (SDS-PAGE). Thus, where cells are radiolabeled prior tothe preparation of cell lysates and passage through the KIAA0175affinity column, proteins having high affinity for KIAA0175, may bedetected by autoradiography. The identity of KIAA0175 specific bindingproteins may be determined by protein sequencing techniques that arereadily available to the skilled artisan, such as Mathews, C. K. et al.,Biochemistry, The Benjamin/Cummings Publishing Company, Inc. pp. 166-170(1990).

Antibodies or Antibody Fragments

KIAA0175 inhibitors of the present invention include antibodies and/orantibody fragments that are effective in reducing KIAA0175 geneexpression and/or biological activity. Suitable antibodies may bemonoclonal, polyclonal or humanized monoclonal antibodies. Antibodiesmay be derived by conventional hybridoma based methodology, fromantisera isolated from KIAA0175 inoculated animals or throughrecombinant DNA technology. Alternatively, inventive antibodies orantibody fragments may be identified in vitro by use of one or more ofthe readily available phage display libraries. Exemplary methods aredisclosed herein.

In one embodiment of the present invention, KIAA0175 inhibitors aremonoclonal antibodies that may be produced as follows. KIAA0175 proteinmay be produced, for example, by expression of KIAA0175 cDNA in abaculovirus based system. By this method, KIAA0175 cDNA or a fragmentthereof is ligated into a suitable plasmid vector that is subsequentlyused to transfect Sf9 cells to facilitate protein production. Inaddition, it may be advantageous to incorporate an epitope tag or othermoiety to facilitate affinity purification of the KIAA0175 protein.Clones of Sf9 cells expressing KIAA0175 are identified, e.g., by enzymelinked immunosorbant assay (ELISA), lysates are prepared and theKIAA0175 protein purified by affinity chromatography and the purifiedprotein is injected, intraperitoneally, into BALB/c mice to induceantibody production. It may be advantageous to add an adjuvant, such asFreund's adjuvant, to increase the resulting immune response.

Serum is tested for the production of specific antibodies and spleencells from animals having a positive specific antibody titer are usedfor cell fusions with myeloma cells to generate hybridoma clones.Supernatants derived from hybridoma clones are tested for the presenceof monoclonal antibodies having specificity against KIAA0175. For ageneral description of monoclonal antibody methodology, See, e.g.,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory (1988).

In addition to the baculovirus expression system, other suitablebacterial or yeast expression systems may be employed for the expressionof KIAA0175 protein or polypeptides thereof. As with the baculovirussystem, it may be advantageous to utilize one of the commerciallyavailable affinity tags to facilitate purification prior to inoculationof the animals. Thus, the KIAA0175 cDNA or fragment thereof may beisolated by, e.g., agarose gel purification and ligated in frame with asuitable tag protein such as 6-His, glutathione-S-transferase (GST) orother such readily available affinity tag. See, e.g., MolecularBiotechnology: Principles and Applications of Recombinant DNA, ASM Presspp. 160-161 (ed. Glick, B. R. and Pasternak, J. J. 1998).

In other embodiments of the present invention, KIAA0175 inhibitors arehumanized anti-KIAA0175 monoclonal antibodies. The phrase “humanizedantibody” refers to an antibody derived from a non-humanantibody—typically a mouse monoclonal antibody. Alternatively, ahumanized antibody may be derived from a chimeric antibody that retainsor substantially retains the antigen-binding properties of the parental,non-human, antibody but which exhibits diminished immunogenicity ascompared to the parental antibody when administered to humans. Thephrase “chimeric antibody,” as used herein, refers to an antibodycontaining sequence derived from two different antibodies (see, e.g.,U.S. Pat. No. 4,816,567) which typically originate from differentspecies. Most typically, chimeric antibodies comprise human and murineantibody fragments, generally human constant and mouse variable regions.

Because humanized antibodies are far less immunogenic in humans than theparental mouse monoclonal antibodies, they can be used for the treatmentof humans with far less risk of anaphylaxis. Thus, these antibodies maybe preferred in therapeutic applications that involve in vivoadministration to a human such as, e.g., use as radiation sensitizersfor the treatment of neoplastic disease or use in methods to reduce theside effects of, e.g., cancer therapy.

Humanized antibodies may be achieved by a variety of methods including,for example: (1) grafting the non-human complementarity determiningregions (CDRs) onto a human framework and constant region (a processreferred to in the art as “humanizing”), or, alternatively, (2)transplanting the entire non-human variable domains, but “cloaking” themwith a human-like surface by replacement of surface residues (a processreferred to in the art as “veneering”). In the present invention,humanized antibodies will include both “humanized” and “veneered”antibodies. These methods are disclosed in, e.g., Jones et al., Nature321:522-525 (1986); Morrison et al., Proc. Natl. Acad. Sci., U.S.A.,81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988);Verhoeyer et al., Science 239:1534-1536 (1988); Padlan, Molec. Immun.28:489-498 (1991); Padlan, Molec. Immunol. 31(3):169-217 (1994); andKettleborough, C. A. et al., Protein Eng. 4(7):773-83 (1991) each ofwhich is incorporated herein by reference.

The phrase “complementarity determining region” refers to amino acidsequences which together define the binding affinity and specificity ofthe natural Fv region of a native immunoglobulin binding site. See,e.g., Chothia et al., J. Mol. Biol. 196:901-917 (1987); Kabat et al.,U.S. Dept. of Health and Human Services NIH Publication No. 91-3242(1991). The phrase “constant region” refers to the portion of theantibody molecule that confers effector functions. In the presentinvention, mouse constant regions are substituted by human constantregions. The constant regions of the subject humanized antibodies arederived from human immunoglobulins. The heavy chain constant region canbe selected from any of the five isotypes: alpha, delta, epsilon, gammaor mu.

One method of humanizing antibodies comprises aligning the non-humanheavy and light chain sequences to human heavy and light chainsequences, selecting and replacing the non-human framework with a humanframework based on such alignment, molecular modeling to predict theconformation of the humanized sequence and comparing to the conformationof the parent antibody. This process is followed by repeated backmutation of residues in the CDR region which disturb the structure ofthe CDRs until the predicted conformation of the humanized sequencemodel closely approximates the conformation of the non-human CDRs of theparent non-human antibody. Such humanized antibodies may be furtherderivatized to facilitate uptake and clearance, e.g., via Ashwellreceptors. See, e.g., U.S. Pat. Nos. 5,530,101 and 5,585,089 whichpatents are incorporated herein by reference.

It will be appreciated that alternative KIAA0175 inhibitor antibodiesmay be readily obtained by other methods commonly known in the art. Oneexemplary methodology for identifying antibodies having a highspecificity for KIAA0175 is the phage display technology.

Phage display libraries for the production of high-affinity antibodiesare described in, for example, Hoogenboom, H. R. et al.,Immunotechnology 4(1):1-20 (1998); Hoogenboom, H. R., Trends Biotechnol.15:62-70 (1997) and McGuinness, B. et al., Nature Bio. Technol.14:1149-1154 (1996) each of which is incorporated herein by reference.Among the advantages of the phage display technology is the ability toisolate antibodies of human origin that cannot otherwise be easilyisolated by conventional hybridoma technology. Furthermore, phagedisplay antibodies may be isolated in vitro without relying on ananimal's immune system.

Antibody phage display libraries may be accomplished, for example, bythe method of McCafferty et al., Nature 348:552-554 (1990) which isincorporated herein by reference. In short, the coding sequence of theantibody variable region is fused to the amino terminus of a phage minorcoat protein (pIII). Expression of the antibody variable region-pIIIfusion construct results in the antibody's “display” on the phagesurface with the corresponding genetic material encompassed within thephage particle.

KIAA0175 protein suitable for screening a phage library may be obtainedby, for example, expression in baculovirus Sf9 cells as described,supra. Alternatively, the KIAA0175 coding region may be PCR amplifiedusing primers specific to the desired region of the KIAA0175 protein.For example, where the inhibitor is directed against KIAA0175's kinasedomain, fragments may be amplified that encode the amino acid sequenceflanking lysine 40 in the active site. Exemplary PCR primers for theamplification of KIAA0175 include, but are not limited to, those setforth in SEQ ID NOs:7 and 8. As discussed above, the KIAA0175 proteinmay be expressed in E. coli or yeast as a fusion with one of thecommercially available affinity tags.

The resulting fusion protein may then be adsorbed to a solid matrix,e.g., a tissue culture plate or bead. Phage expressing antibodies havingthe desired anti-KIAA0175 binding properties may subsequently beisolated by successive panning, in the case of a solid matrix, or byaffinity adsorption to a KIAA0175 antigen column. Phage having thedesired KIAA0175 inhibitory activities may be reintroduced into bacteriaby infection and propagated by standard methods known to those skilledin the art See Hoogenboom, H. R., Trends Biotechnol., supra for a reviewof methods for screening for positive antibody-pIII phage.

Small Molecules

The present invention also provides small molecule KIAA0175 inhibitorsthat may be readily identified through routine application ofhigh-throughput screening (HTS) methodologies. Reviewed by Persidis, A.,Nature Biotechnology 16:488-489 (1998). HTS methods generally refer tothose technologies that permit the rapid assaying of lead compounds,such as small molecules, for therapeutic potential. HTS methodologyemploys robotic handling of test materials, detection of positivesignals and interpretation of data. Such methodologies include, e.g.,robotic screening technology using soluble molecules as well ascell-based systems such as the two-hybrid system described in detailabove.

A variety of cell line-based HTS methods are available that benefit fromtheir ease of manipulation and clinical relevance of interactions thatoccur within a cellular context as opposed to in solution. Leadcompounds may be identified via incorporation of radioactivity orthrough optical assays that rely on absorbance, fluorescence orluminescence as read-outs. See, e.g., Gonzalez, J. E. et al., Curr.Opin. Biotechnol. 9(6):624-631 (1998) incorporated herein by reference.

HTS methodology may be employed, e.g., to screen for lead compounds thatblock one of KIAA0175's biological activities such as itsautophosphorylation activity. By this method, KIAA0175 protein may beimmunoprecipitated from cells expressing the protein and applied towells on an assay plate suitable for robotic screening. Individual testcompounds may then be contacted with the immunoprecipitated protein andthe effect of each test compound on KIAA0175 kinase activity assessedby, e.g., incubating in the presence of γ-³²P-ATP in a suitable buffersystem, see Example 2, and measuring the incorporation of ³²P.

Methods for Assessing the Efficacy of KIAA0175 Inhibitors

Lead molecules or compounds, whether anti-sense molecules or ribozymes,proteins and/or peptides, antibodies and/or antibody fragments or smallmolecules, that are identified either by one of the methods describedherein or via techniques that are otherwise available in the art, may befurther characterized in a variety of in vitro, ex vivo and in vivoanimal model assay systems for their ability to inhibit KIAA0175 geneexpression or biological activity. As discussed in further detail in theExamples provided below, KIAA0175 inhibitors of the present inventionare effective in reducing not only KIAA0175 expression levels but also,inter alia, (1) in decreasing intracellular p53 and/or p21 proteinlevels in the target mammalian cell; (2) in reducing cell cycle phasetransitions; and (3) in increasing cells to chemotherapy and/orradiation sensitizers. Thus, the present invention further disclosesmethods that permit the skilled artisan to assess the effect ofcandidate inhibitors on each of these three parameters.

As noted above and as presented in the Examples provided herein, supra,candidate KIAA0175 inhibitors may be tested by administration to cellsthat either express endogenous KIAA0175 or that are made to expressKIAA0175 by transfection of a mammalian cell with a recombinant KIAA0175plasmid construct. Suitable cell lines for this purpose include, e.g.,the HT1080 and HCT116 cell lines both of which express relatively highlevels of endogenous KIAA0175. While the utility of these cell lines isspecifically disclosed herein, it will be apparent to one of skill inthe art that alternative cell lines may be used in accordance with themethodologies disclosed herein the choice of which cell line willdepend, for example, on the specific application contemplated.

Effective KIAA0175 inhibitory molecules will be effective in reducingthe levels of KIAA0175 mRNA as determined, e.g., by Northern blot orRT-PCR analysis. See Example 4; for a general description of theseprocedures, see, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual Cold Spring Harbor Press (1989) and Molecular Biotechnology:Principles and Applications of Recombinant DNA, ASM Press (ed. Glick, B.R. and Pasternak, J. J. 1998) incorporated herein by reference. Theeffectiveness of a given candidate anti-sense molecule may be assessedby comparison with a control “anti-sense” molecule known to have nosubstantial effect on KIAA0175 expression when administered to amammalian cell. Exemplary control molecules include the AKT and FITCprimers disclosed in Example 4.

Putative KIAA0175 inhibitory anti-sense molecules may be additionallycharacterized for their effect on p53 and p21 protein levels whenadministered to a mammalian cell. Thus, for example, such candidatemolecules may be administered to HT1080 or HCT116 cells. Following asuitable period of culture, the transfected cells may additionally bechallenged with γ-irradiation. The cellular protein levels of p53 andp21 may be assessed either before irradiation or at various time-pointsafter challenge, e.g., by Western analysis using antibodies havingspecificity to each of these proteins. See Examples 5 and 6 and, e.g.,Sambrook et al., supra. Levels of a suitable negative control protein,such as Erk2, may be analyzed in order to verify the specificity of eachcandidate KIAA0175 inhibitory anti-sense molecule. Effective KIAA0175inhibitory anti-sense molecules may cause a reduction in either or bothof p53 and p21 protein levels. As well, KIAA0175 inhibitory anti-sensemolecules used as radiation sensitizers, see infra, will also reduce theγ-irradiation induced increase in p53 and/or p21 protein levels.

In alternate embodiments of the present invention, the effect ofKIAA0175 inhibitors on the rate of DNA synthesis after challenge with aradiation or chemotherapeutic agent may be assessed by, e.g., the methodof Young and Painter. Hum. Genet. 82:113-117 (1989). Briefly, culturecells may be incubated in the presence of ¹⁴C-thymidine prior toexposure to, e.g., X-rays. Immediatedly after irradiation, cells areincubated for a short period prior to addition of ³H-thymidine. Cellsare washed, treated with perchloric acid and filtered (Whatman GF/C).The filters are rinsed with perchloric acid, 70% alcohol and then 100%ethanol; radioactivity is measured and the resulting ³H/¹⁴C ratios usedto determine the rates of DNA synthesis.

KIAA0175 inhibitors effective in reducing KIAA0175 gene expressionand/or p21 or p53 protein levels by one or more of the methods discussedabove may be further characterized in vivo for efficacy in one of thereadily available animal model systems. The various animal model systemsfor study of cancer and genetic instability associated genes have beenthe subject of a recent review. Donehower, L. A. Cancer Surveys29:329-352 (1997) incorporated herein by reference.

By still further embodiments, KIAA0175 inhibitors may be identified byassessing the effect of candidate inhibitors on cell cycle arrest inresponse to γ-irradiation as disclosed herein, infra, in Example 7.

KIAA0175 Inhibitors as Chemotherapy and Radiation Sensitizers

As discussed above, the present invention provides KIAA0175 inhibitorsas well as compositions and methods employing KIAA0175 inhibitors thatare useful as chemotherapy and radiation sensitizers for, inter alia,the treatment of neoplastic disease. As used herein, the term“sensitizer” generally refers to the property of a molecule orcomposition to induce in a cell hypersensitivity to a chemotherapeuticdrug, to ionizing radiation and/or to other DNA-damaging agents.Exemplary methods employ gene delivery techniques, as described indetail, infra, to administer KIAA0175 inhibitory anti-sense molecules totarget cells. See, also, Example 8.

The methods provided herein share the common feature thathypersensitization is achieved by administration of one or more KIAA0175inhibitors. Within some embodiments, chemotherapy and/or radiationhypersensitivity is achieved by administration of an anti-sensemolecule. In certain other embodiments, chemotherapy and/orradiosensitizing anti-sense molecules may be linked to aradiation-inducible promoter to localize expression by externalradiation beams. See, e.g., McBride, W. H. et al., Nat. Med. 1:1215-1217(1995) and Weichselbaum, R. R. et al., Cancer Res. 54:4266-4269 (1994)(describing the combination of gene delivery and radiation as a paradigmfor radiotherapy). By alternate embodiments, expression of the KIAA0175inhibitory anti-sense molecule may be restricted by use of atissue-specific or cell cycle-specific promoter. Vile, R. G. et al.,Cancer Res. 53:962-967 (1993) and Vile, R. G., Semin. Cancer Biol. 5:437-443 (1994) (discussing tumor specific targeted gene expression).Other embodiments of the present invention employ trophic viruses thatare confined to particular organs or structures. Each of these exemplarymethods is incorporated herein by reference.

By the present methods, beneficial improvement of chemo and/orradiotherapy will be achieved despite low transfection efficiency and/ortransient gene expression. This will be the case especially where thegene-delivery methodology is repeated throughout the course of therapy.Transfection efficiencies are expected to be in the order of 10% to 70%.

Use of KIAA0175 Inhibitors to Reduce the Severity of Cancer Therapy SideEffects

Conventional cancer treatment regimens, including both chemotherapy andradiation therapy, frequently have severe side effects that compromisetheir efficacy. These side effects result, in part, from p53-mediatedapoptosis of treated cells. Thus, it has been suggested that suppressionof p53 activity may be effective in reducing apoptosis and as aconsequence decreasing the adverse side effects associated with cancertherapy. Komarov, P. G. et al., Science 285:1733-1737 (1999)incorporated herein by reference.

High levels of p53 have been detected in a variety of normal tissues,including lymphoid and hematopoietic organs, intestinal epithelia, andthe testis that are damaged by anticancer treatments. Rogel, A. et al.,Mol. Cell. Biol. 5:2851 (1985); Schmidt, P. et al., Development 113:857(1991); Schwartz, D. et al., Oncogene 8:1487 (1993) and Komarova, E. A.et al., EMBO J. 16:1391 (1997). In addition, p53-dependent apoptosisoccurs in these sensitive tissues shortly after γ-irradiation. Komarova,id.; Hendry, J. H. et al., Int. J. Radiat. Biol. 70:677 (1996); andTron, V. A. et al., Am. J. Pathol. 153:579 (1998). Thus, it has beenproposed that p53 may be an appropriate target for therapeuticsuppression thereby reducing damage to normal cells. Komarova, E. A. etal., Semin. Cancer Biol. 8:389 (1998).

It has been discovered, as part of the present invention, that KIAA0175inhibitors are effective in reducing the intracellular levels of p53protein. Accordingly, KIAA0175 inhibitors may be effective as drugs forreducing the side effects of cancer therapy and other types of stressassociated with p53 induction.

Lead compounds may be identified, by the methods provided herein or byother suitable methods available in the art, that are effective inreducing p53 protein levels when administered to a mammalian cell. Forexample, as disclosed in Example 5, KIAA0175 inhibitors may beadministered to HT1080 or HCT116 cells and the resulting levels of p53may be assessed by Western analysis using p53-specific antibodies.KIAA0175 inhibitors that are effective in reducing p53 expression levelsmay be further tested, by the method of Komarov, P. G. et al., supra, ina conventional model of p53-dependent apoptosis such as the mouse cellline C8. Lowe, S. W. et al., Cell 74:957 (1993) incorporated herein byreference (describing the C8 cell-line as a mouse embryo fibroblasttransformed with E1a+ras that undergoes rapid p53-dependent apoptosis inresponse to a variety of treatments).

The in vivo efficacy of KIAA0175 inhibitors found to be active inreducing p53-mediated cellular apoptosis may be further characterized ina suitable animal model system. For example, two different strains ofmice, i.e., C57BL or Balb/c, may be treated with lethal and sublethaldoses of whole-body γ-irradiation after administration of one or moreKIAA0175 inhibitor. See Komarov, P. G. et al., supra.

Administration of KIAA0175 Inhibitors and Compositions Thereof

The present invention provides KIAA0175 inhibitors and compositionscomprising one or more KIAA0175 inhibitor as well as methods that employthese inventive inhibitors in in vivo, ex vivo, and in vitroapplications where it is advantageous to reduce or eliminate theexpression or activity of KIAA0175 or a functionally downstream moleculesuch as p53 or p21. As indicated above, KIAA0175 inhibitor basedcompositions will find utility in the treatment of neoplastic diseaseand related conditions where treatment regimens are improved byradiation hypersensitivity of tumor cells. Alternatively, KIAA0175inhibitors may find use as drugs for reducing the side effects of, e.g.,cancer therapeutics and other agents that cause p53-mediated cell stressand apoptosis.

Compositions may be administered parenterally, topically, orally orlocally for therapeutic treatment. Preferably, the compositions areadministered orally or parenterally, i.e., intravenously,intraperitoneally, intradermally or intramuscularly.

Inventive compositions will include one or more KIAA0175 inhibitor andmay further comprise a pharmaceutically acceptable carrier or excipient.A variety of aqueous carriers may be used, e.g., water, buffered water,0.4% saline, 0.3% glycine and the like, and may include other proteinsfor enhanced stability, such as albumin, lipoprotein, globulin, etc.,subjected to mild chemical modifications or the like.

Inhibitors of the present invention may be combined with additional oralternative inhibitors of, for example, ATM, DNA-PK or ATR to achievestill greater levels of radiation hypersensitivity. See, e.g., Westpahlet al., Nat. Genet. 16:397 (1997) incorporated herein by reference.

KIAA0175 inhibitors useful as radiation sensitizers or otherwise usefulin the treatment of disease in mammals will often be preparedsubstantially free of other naturally occurring immunoglobulins or otherbiological molecules. Preferred KIAA0175 inhibitors will also exhibitminimal toxicity when administered to a mammal.

The compositions of the invention may be sterilized by conventional,well known sterilization techniques. The resulting solutions may bepackaged for use or filtered under aseptic conditions and lyophilized,the lyophilized preparation being combined with a sterile solution priorto administration. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents and the like, for example, sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride andstabilizers (e.g., 1-20% maltose, etc.).

The selection of the appropriate method for administering KIAA0175inhibitors of the present invention will depend on the nature of theapplication envisioned as well as the nature of the KIAA0175 inhibitor.Thus, for example, the precise methodology for administering a KIAA0175inhibitor will depend upon whether it is an anti-sense molecule, aprotein and/or peptide, an antibody or antibody fragment or a smallmolecule. Other considerations include, for example, whether theKIAA0175 inhibitor will be used to increase radiation hypersensitivityor to reduce the side effects of cancer therapeutics.

A variety of methods are available in the art for the administration ofanti-sense molecules. Exemplary methods include gene deliverytechniques, including both viral and non-viral based methods as well asliposome mediated delivery methods

Gene delivery methodologies will be effective to, for example, sensitizetumor cells to irradiation and/or chemotherapeutic drugs. See, Wheldon,T. E. et al., Radiother Oncol 48(1):5-13 (1998) (gene deliverymethodologies for enhancement of fractionated radiotherapy). By thesemethodologies, substantial therapeutic benefit may be achieved despitetransfection efficiencies significantly less than 100%, transientretention of the transfected inhibitor and/or existence of asubpopulation of target cells refractory to therapy.

As discussed above, the limitation of conventional radiotherapy is thatits tumor specificity is weak. This limitation may be overcome by directtargeting of the expression of anti-sense KIAA0175 inhibitors to tumorcells. This may be achieved, for example, by linking the KIAA0175inhibitor gene to a radiation-inducible promoter so that gene therapycan be localized by external radiation beams. McBride, W. H. et al.,Nat. Med. 1:1215-1217 (1995) and Weichselbaum, R. R. et al., LancetSuppl II:S10-S12 (1997) each of which is incorporated herein byreference.

Alternatively, gene delivery methodology may be used to directlyknock-out endogenous KIAA0175 within tumor cells thereby rendering thetumor cells hypersensitive to radiation and chemotherapeutic drugs. Forexample, the KIAA0175 gene may be targeted by transfection of a genedelivery vector carrying a KIAA0175 inhibitor. Preferential transfectioninto or expression within tumor cells may be achieved through use of atissue-specific or cell cycle-specific promoter, such as, e.g.,promoters for prostate-specific antigen or for immunoglobulin genes(Vile, R. G. et al., Cancer Res. 53:962-967 (1993) and Vile, R. G.,Semin. Cancer Biol. 5:437-443 (1994)) or through the use of trophicviruses that are confined to particular organs or structures, such as,e.g., a replication selective and neurotrophic virus that can onlyinfect proliferating cells in the central nervous system.

Thus, to achieve therapeutic benefit, KIAA0175 within the tumor cellsshould be preferentially inhibited. This can be accomplished bytransfecting a gene expressing a KIAA0175 inhibitor, a KIAA0175anti-sense molecule, a KIAA0175 gene specific repressor, or an inhibitorof the protein product of the KIAA0175 gene.

As used herein, the phrase “gene delivery vector” refers generally to anucleic acid construct that carries and, within certain embodiments, iscapable of directing the expression of an anti-sense molecule ofinterest, as described in, for example, Molecular Biotechnology:Principles and Applications of Recombinant DNA, Ch. 21, pp. 555-590 (ed.B. P. Glick and J. J. Pasternak, 2^(nd) ed. 1998); Jolly, Cancer GeneTher. 1:51-64 (1994); Kimura, Human Gene Ther. 5:845-852 (1994);Connelly, Human Gene Ther. 6:185-193 (1995); and Kaplitt, Nat. Gen.6:148-153 (1994).

A number of virus and non-virus based gene delivery vector systems havebeen described that are suitable for the administration of KIAA0175inhibitors. Virus based gene delivery systems include, but are notlimited to retrovirus, such as Moloney murine leukemia virus,spumaviruses and lentiviruses; adenovirus; adeno-associated virus; andherpes-simplex virus vector systems. Viruses of each type are readilyavailable from depositories or collections such as the American TypeCulture Collection (ATCC; 10801 University Boulevard, Manassas, Va.20110-2209) or may be isolated from known sources using commonlyavailable materials and techniques.

The gene delivery vector systems of the present invention will findapplications both in in vivo as well as ex vivo therapeutic regimens.Each of these applications is described in further detail below.

1. Retroviral Gene Delivery Vector Systems

Within one aspect of the present invention, retroviral gene deliveryvectors are provided that are constructed to carry or express a KIAA0175inhibitory anti-sense molecule. As used herein, the term “KIAA0175inhibitory anti-sense molecule” refers generally to a nucleic acidsequence having KIAA0175 inhibitory activity. More specifically, suchanti-sense molecules will reduce KIAA0175 gene expression as well as p53and/or p21 protein levels. Retroviral gene delivery vectors of thepresent invention may be readily constructed from a wide variety ofretroviruses, including for example, B, C, and D type retroviruses aswell as spumaviruses and lentiviruses. See RNA Tumor Viruses, ColdSpring Harbor Laboratory (2^(nd) ed. 1985).

Any of the above retroviruses may be readily utilized in order toassemble or construct retroviral gene delivery vectors given thedisclosure provided herein, and standard recombinant DNA techniques.See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press (2d ed. 1989) and Kunkle, Proc. Natl.Acad. Sci. U.S.A. 82:488 (1985). In addition, within certain embodimentsof the invention, portions of the retroviral gene delivery vectors maybe derived from different retroviruses.

A retroviral vector, suitable for the expression of a KIAA0175inhibitory anti-sense molecule, must include at least onetranscriptional promoter/enhancer or locus defining element(s), or otherelements that control gene expression by other means such as alternatesplicing, nuclear RNA export, post-translational modification ofmessenger, or post-transcriptional modification of protein. Such vectorconstructs must also include a packaging signal, long terminal repeats(LTRs) or portion thereof, and positive and negative strand primerbinding sites appropriate to the retrovirus used (if these are notalready present in the retroviral vector). Optionally, the retroviralvector may also include a signal that directs polyadenylation,selectable markers such as Neomycin resistance, TK, hygromycinresistance, phleomycin resistance histidinol resistance, or DHFR, aswell as one or more restriction sites and a translation terminationsequence. Within one aspect of the present invention, retroviral genedelivery vector constructs are provided comprising a 5′ LTR, a tRNAbinding site, a packaging signal, one or more heterologous sequences, anorigin of second strand DNA synthesis and a 3′ LTR, wherein the vectorconstruct lacks gag/pol or env coding sequences.

Other retroviral gene delivery vectors may likewise be utilized withinthe context of the present invention, including, for example, thosedisclosed in the following each of which is incorporated herein byreference: EP 0,415,731; WO 90/07936; WO 94/03622; WO 93/25698; WO93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO 93/10218; Vile etal., Cancer Res. 53:3860-3864 (1993); Vile et al., Cancer Res.53:962-967 (1993); Ram et al., Cancer Res. 53:83-88 (1993); Takamiya etal., J. Neurosci. Res. 33:493-503 (1992); Baba et al., J. Neurosurg.79:729-735 (1993); U.S. Pat. No. 4,777,127, GB 2,200,651, EP 0,345,242and WO 91/02805.

Packaging cell lines suitable for use with the above describedretroviral gene delivery vector constructs may be readily prepared. See,e.g., U.S. Pat. Nos. 5,716,832 and 5,591,624. These packaging cell linesmay be utilized to create producer cell lines (also termed vector celllines or “VCLs”) for the production of recombinant vector particles. Itmay be preferred to use packaging cell lines made from human (e.g.,HT1080 cells) or mink parent cell lines, thereby allowing production ofrecombinant retroviruses that avoid inactivation in human serum.

2. Adeno-Associated Viral Gene Delivery Vector Systems

Adeno-associated viruses (AAV) possess a number of qualities that makethem particularly suitable for the development of gene delivery vectorsgenerally and for the delivery of polynucleotides encoding KIAA0175inhibitory anti-sense molecules in particular. For a general review ofAAV expression systems, see Rabinowitz et al., Current Opin. Biotech.9(5):470-475 (1998). AAV is a non-pathogenic, defective human parvovirusthat is non-infective without an adeno or herpes helper virus. Thus, inthe absence of a helper virus, AAV becomes integrated latently into thehost genome. In addition, AAV has the advantage over the retroviruses,discussed above, in being able to transduce a wide range of bothdividing and quiescent cell types.

A variety of AAV gene delivery vectors may be utilized to direct theexpression of one or more KIAA0175 inhibitor anti-sense molecule.Representative examples of such vectors include the AAV vectorsdisclosed by Srivastava in WO 93/09239; Samulski, et al. J. Virol.63:3822-3828 (1989); Mendelson, et al. Virol. 166:154-165 (1988); andFlotte, et al. Proc. Natl. Acad. Sci. USA. 90(22):10613-10617 (1993)incorporated herein by reference.

Briefly, an AAV gene delivery vector of the present invention mayinclude, in order, a 5′ adeno-associated virus inverted terminal repeat;a polynucleotide encoding the KIAA0175 inhibitory anti-sense molecule; asequence operably linked to the KIAA0175 inhibitory anti-sense moleculethat regulates its expression in a target tissue, organ or cell; and a3′ adeno-associated virus inverted terminal repeat. A suitableregulatory sequence for the expression of KIAA0175 inhibitory anti-sensemolecule is, e.g., the enhancer/promoter sequence of cytomegalovirus(CMV). In addition, the AAV vector may preferably have a polyadenylationsequence such as the bovine growth hormone (BGH) polyadenylationsequence. Generally, AAV vectors should have one copy of the AAV ITR ateach end of the KIAA0175 inhibitory anti-sense molecule, to allowreplication, packaging, efficient integration into the host cell genomeand rescue from the chromosome. The 5′ ITR sequence consists ofnucleotides 1 to 145 at the 5′ end of the AAV DNA genome, and the 3′ ITRincludes nucleotides 4681 to 4536 of the AAV genome. Preferably, the AAVvector may also include at least 10 nucleotides following the end of theITR (i.e., a portion of the so-called “D region”).

Optimal packaging of an adeno-associated virus gene delivery vectorrequires that the 5′ and 3′ ITRs be separated by approximately 2-5 kb.It will be apparent, however, that the ideal spacing between ITRsequences may vary depending on the particular packaging systemutilized. This spacing may be achieved by incorporating a “stuffer” or“filler” polynucleotide fragment to bring the total size of the nucleicacid sequence between the two ITRs to between 2 and 5 kb. Thus, wherethe KIAA0175 inhibitory anti-sense molecule is smaller than 2-5 kb, anon-coding stuffer polynucleotide may be incorporated, for example, 3′to the 5′ ITR sequence and 5′ of the KIAA0175 inhibitory anti-sensemolecule. The precise nucleotide sequence of the stuffer fragment is notan essential element of the final construct.

Depending upon the precise application contemplated, rather thanincorporating a stuffer fragment, multiple copies of the KIAA0175inhibitory anti-sense molecule may be inserted, inter alia, to achievethe optimal ITR sequence spacing. It may be preferred to organize thepolynucleotides as two or more separate transcription units each withits own promoter and polyadenylation signal.

Recombinant AAV vectors of the present invention may be generated from avariety of adeno-associated viruses, including for example, serotypes 1through 6. For example, ITRs from any AAV serotype are expected to havesimilar structures and functions with regard to replication,integration, excision and transcriptional mechanisms.

Within certain embodiments of the invention, expression of the KIAA0175inhibitory anti-sense molecule may be accomplished by a separatepromoter (e.g., a viral promoter). Representative examples of suitablepromoters in this regard include a CMV promoter, an RSV promoter, anSV40 promoter, or a MoMLV promoter. Other promoters that may similarlybe utilized within the context of the present invention include cell ortissue specific promoters or inducible promoters. Representativeinducible promoters include tetracycline-response promoters (e.g., the“Tet” promoter) as described in Gossen et al., Proc. Natl. Acad. Sci.U.S.A. 89:5547-5551 (1992); Gossen et al., Science 268:1766-1769 (1995);Baron et al., Nucl. Acids Res. 25:2723-2729 (1997); Blau et al., Proc.Natl. Acad. Sci. USA. 96:797-799 (1999); Bohl et al., Blood 92:1512-1517(1998); and Haberman et al., Gene Therapy 5:1604-1611 (1998); theecdysone promoter system as described in No et al., Proc. Natl. Acad.Sci. USA. 93:3346-3351 (1996); and other regulated promoters or promotersystems as described in Rivera et al., Nat. Med. 2:1028-1032 (1996).

The AAV gene delivery vector may also contain additional sequences, forexample from an adenovirus, which assist in effecting a desired functionfor the vector. Such sequences include, for example, those which assistin packaging the AAV gene delivery vector in adenovirus particles.

Packaging cell lines suitable for producing adeno-associated viralvectors may be routinely prepared given readily available techniques.See, e.g., U.S. Pat. No. 5,872,005, incorporated herein by reference. Ata minimum, suitable packaging systems for AAV gene delivery systems ofthe present invention will include the AAV replication and capsid genes.

Preferred packaging cell lines may contain both an AAV helper virus aswell as an AAV gene delivery vector containing the KIAA0175 inhibitoryanti-sense molecule. For detailed descriptions of representativepackaging cell line systems, see, e.g. Holscher, C. et al., J. Virol.68:7169-7177 (1994); Clark, K. R. et al., Hum. Gene Ther. 6:1329-1341(1995); and Tamayosa, K et al., Hum. Gen. Ther. 7:507-513 (1996) whichare incorporated herein by reference.

Alternatively, packaging of AAV may be achieved in vitro in a cell freesystem to obviate transfection protocols or packaging cell lines. Suchin vitro systems incorporate an AAV gene delivery vector bearing theKIAA0175 inhibitory anti-sense molecule and a source of Rep-protein,capsid-protein and Adenovirus proteins that supply helper-viralfunctions. The latter proteins are typically supplied in the form of acell extract. Representative in vitro systems are further described inDing, L. et al., Gen. Ther. 4:1167-1172 (1997) and Zhou, Z. et al., J.Virol. 72:3241-3247 (1998) which are incorporated herein by reference.

3. Other Viral Gene Delivery Vector Systems

In addition to retroviral vectors and adeno-associated virus-basedvectors, numerous other viral gene delivery vector systems may also beutilized for the expression of KIAA0175 inhibitory anti-sense molecules.For example, within one embodiment of the invention adenoviral vectorsmay be employed. Representative examples of such vectors include thosedescribed by, for example, Berkner, Biotechniques 6:616-627 (1988);Rosenfeld et al., Science 252:431-434 (1991); WO 93/9191; Kolls et al.,Proc. Natl. Acad. Sci. U.S.A. 91(1)215-219 (1994); Kass-Eisler et al.,Proc. Natl. Acad. Sci. U.S.A. 90(24):11498-502 (1993); Guzman et al.,Circulation 88(6:2838-48 (1993); Guzman et al., Cir. Res.73(6):1202-1207 (1993); Zabner et al., Cell 75(2):207-216 (1993); Li etal., Hum. Gene Ther. 4 :403-409 (1993); Caillaud et al., Eur. J.Neurosci. 5(10):1287-1291 (1993); Vincent et al., Nat. Genet.5(2):130-134 (1993); Jaffe et al., Nat. Genet. 1(5):372-378 (1992); andLevrero et al., Gene 101(2):195-202 (1991); and WO 93/07283; WO93/06223; and WO 93/07282.

Gene delivery vectors of the present invention also include herpesvectors. Representative examples of such vectors include those disclosedby Kit in Adv. Exp. Med. Biol. 215:219-236 (1989); and those disclosedin U.S. Pat. No. 5,288,641 and EP 0176170 (Roizman). Additionalexemplary herpes simplex virus vectors include HFEM/ICP6-LacZ disclosedin WO 95/04139 (Wistar Institute), pHSVlac described in Geller, Science241:1667-1669 (1988), and in WO 90/09441 and WO 92/07945; HSVUs3::pgC-lacZ described in Fink, Human Gene Therapy 3:11-19 (1992); andHSV 7134, 2 RH 105 and GAL4 described in EP 0453242 (Breakefield), andthose deposited with the ATCC as accession numbers ATCC VR-977 and ATCCVR-260.

Gene delivery vectors may also be generated from a wide variety of otherviruses including, for example, poliovirus (Evans et al., Nature339:385-388 (1989); and Sabin, J. Biol. Standardization 1:115-118(1973)); rhinovirus; pox viruses, such as canary pox virus or vacciniavirus (Fisher-Hoch et al., Proc. Natl. Acad. Sci. U.S.A. 86:317-321(1989); Flexner et al., Ann. N.Y. Acad. Sci. 569:86-103 (1989); Flexneret al., Vaccine 8:17-21 (1990); U.S. Pat. Nos. 4,603,112, 4,769,330 and5,017,487; WO 89/01973); SV40 (Mulligan et al., Nature 277:108-114(1979); influenza virus (Luytjes et al., Cell 59:1107-1113 (1989);McMicheal et al., N. Eng. J. Med. 309:13-17 (1983); and Yap et al.,Nature 273:238-239 (1978)); HIV (Poznansky, J. Virol. 65:532-536(1991)); measles (EP 0 440,219); astrovirus (Munroe et al., J. Vir.67:3611-3614 (1993)); and coronavirus, as well as other viral systems(e.g., EP 0,440,219; WO 92/06693; U.S. Pat. No. 5,166,057).

4. Non-viral Gene Delivery Vectors

Other gene delivery vectors and methods that may be employed for theexpression of KIAA0175 inhibitory anti-sense molecules such as, forexample, nucleic acid expression vectors; polycationic condensed DNAlinked or unlinked to killed adenovirus alone, for example, see Curiel,Hum Gene Ther 3:147-154 (1992); ligand linked DNA, for example, see Wu,J Biol Chem 264:16985-16987 (1989); eucaryotic cell delivery vectors;deposition of photopolymerized hydrogel materials; hand-held genedelivery particle gun, as described in U.S. Pat. No. 5,149,655; ionizingradiation as described in U.S. Pat. No. 5,206,152 and in WO 92/11033;nucleic charge neutralization or fusion with cell membranes. Additionalapproaches are described in Philip, Mol Cell Biol 14:2411-2418 (1994),and in Woffendin, Proc. Natl. Acad. Sci. 91:1581-1585 (1994).

Particle mediated gene delivery may be employed. Briefly, the KIAA0175inhibitory anti-sense molecule of interest can be inserted intoconventional vectors that contain conventional control sequences forhigh level expression, and then be incubated with synthetic genedelivery molecules such as polymeric DNA-binding cations likepolylysine, protamine, and albumin, linked to cell targeting ligandssuch as asialoorosomucoid, as described in Wu, et al., J. Biol. Chem.262:4429-4432 (1987), insulin as described in Hucked, Biochem Pharmacol40:253-263 (1990), galactose as described in Plank, Bioconjugate Chem3:533-539 (1992), lactose or transferrin.

Naked DNA may also be employed. Exemplary naked DNA introduction methodsare described in WO 90/11092 and U.S. Pat. No. 5,580,859. Uptakeefficiency may be improved using biodegradable latex beads. DNA coatedlatex beads are efficiently transported into cells after endocytosisinitiation by the beads. The method may be improved further by treatmentof the beads to increase hydrophobicity and thereby facilitatedisruption of the endosome and release of the DNA into the cytoplasm.

Liposomes that can act as gene delivery vehicles are described in U.S.Pat. No. 5,422,120, PCT Patent Publication Nos. WO 95/13796, WO94/23697, and WO 91/144445, and European Patent Publication No. 524,968.Nucleic acid sequences can be inserted into conventional vectors thatcontain conventional control sequences for high level expression, andthen be incubated with synthetic gene delivery molecules such aspolymeric DNA-binding cations like polylysine, protamine, and albumin,linked to cell targeting ligands such as asialoorosomucoid, insulin,galactose, lactose, or transferrin. Other delivery systems include theuse of liposomes to encapsulate DNA comprising the gene under thecontrol of a variety of tissue-specific or ubiquitously-activepromoters. Further non-viral delivery suitable for use includesmechanical delivery systems such as the approach described in Woffendinet al., Proc. Natl. Acad. Sci. USA. 91(24):11581-11585 (1994). Moreover,the coding sequence and the product of expression of such can bedelivered through deposition of photopolymerized hydrogel materials.

Exemplary liposome and polycationic gene delivery vehicles are thosedescribed in U.S. Pat. Nos. 5,422,120 and 4,762,915, in PCT PatentPublication Nos. WO 95/13796, WO 94/23697, and WO 91/14445, in EuropeanPatent Publication No. 524,968 and in Starrier, Biochemistry, pp.236-240 (1975) W.H. Freeman, San Francisco; Shokai, Biochem. Biophys.Acta. 600:1 (1980); Bayer, Biochem. Biophys. Acta. 550:464 (1979);Rivet, Methods Enzymol. 149:119 (1987); Wang, Proc. Natl. Acad. Sci.U.S.A. 84:7851 (1987); Plant, Anal. Biochem. 176:420 (1989).

“Therapeutically effective amount” as used herein, is the precise amountof the compositions of the present invention to be administered and canbe determined by a physician with consideration of individualdifferences in age, weight, tumor type and size, or extent ofmetastasis, and condition of the patient. It can generally be statedthat a pharmaceutical composition comprising the subject inhibitors istherapeutically effective when any effect of the inhibitors describedherein is achieved (e.g. inhibition of KIAA0175 expression levels, ofp53, p21, or other molecules downstream of KIAA0175), when the desiredsensitization of tumor cells to irradiation or to chemotherapeutic drugsis achieved, and/or when the side effects of cancer therapeutics arereduced. The optimal dosage and treatment regime for a particularpatient can readily be determined by one skilled in the art of medicineby monitoring the patient for signs of disease or improvement andadjusting the treatment accordingly.

EXAMPLES

The following experimental examples are offered by way of illustration,not limitation.

Example 1 Expression of KIAA0175 mRNA

This example discloses Northern blot analysis of the tissue- and cancercell-specific expression of KIAA0175 mRNA.

A KIAA0175 specific DNA probe was generated from a 0.7 kb internalfragment of the KIAA0175 open reading frame. This DNA fragment wasradiolabeled with α-³²P-ATP by standard methods (Stratagene; La Jolla,Calif.) and was used to probe commercially available tissue- and cancercell-line blots obtained from Clontech (7760-1, 7759-1, and 7757-1).Specific hybridization of the KIAA0175 probe was detected byautoradiography.

Of the various tissues tested by Northern blot analysis, testisexpressed the highest level of KIAA0175 mRNA. Thymus and colon expresseda lower level of KIAA0175 mRNA followed, in order of decreasingexpression, by placenta and spleen. Of the various cancer cell linesexamined, MOLT-4 showed the highest levels of KIAA0175 mRNA followed byHeLa S3, K562, and SW480 cells, that expressed intermediate levels ofKIAA0175 mRNA, and HL-60 and Raji cells that expressed still lowerlevels of KIAA0175 mRNA. KIAA0175 mRNA in A549 and G361 cells was barelydetectable.

Example 2 Autophosphorylation of the KIAA0175 Protein

This example discloses that KIAA0175 possesses an autophosphorylationactivity and that this activity may be eliminated by a single amino acidsubstitution at lysine-40.

Plasmid constructs expressing the KIAA0175 wild-type and the kinaseinactive K40A mutant protein were prepared. These plasmids express bothKIAA0175 variants as hemagglutinin (HA) fusion proteins therebyfacilitating their isolation via immunoprecipitation and detectionthrough Western hybridization.

The plasmid vector pcDNA3.1 (Invitrogen) contains a multiple cloningsite and CMV promoter to facilitate the subcloning and expression theKIAA0175 cDNAs having an HA tag sequence.

Cos 7 cells were transfected with either the naked vector pcDNA3.1 orthe pcDNA3.1 expressing the wild-type or kinase inactive KIAA0175proteins. The KIAA0175 proteins were immunoprecipitated with anti-HAmonoclonal antibody (COVANCE/Babco). Kinase activity was determined byincubating the immunoprecipitated proteins in the presence of 10 μCiγ-³²P-ATP, 10 mM ATP, 1 mM DTT, 10 mM MgCl₂, 10 mM MnCl₂ and 50 mM Tris,pH7.5. Proteins were subjected to electrophoresis on an SDSpolyacrylamide gel and the proteins were transferred to a nitrocellulosemembrane. Kinase activity was determined by measuring ³²P incorporationas detected by autoradiography. Immunoprecipitated protein was detectedby Western analysis with anti-HA antibodies.

The data revealed that Cos 7 cells transfected with the naked pcDNA3.1plasmid vector do not produce HA precipitable KIAA0175 protein. Cos 7cells transfected with either the wild-type or the K40A mutant produceapproximately equivalent amounts of the respective KIAA0175 protein butonly the wild-type protein has significant kinase activity.

Example 3 Recombinant and Endogenous KIAA0175 Protein Levels AfterExposure of Cells to γ-Irradiation and Hydroxyurea

This Example discloses that KIAA0175 protein levels increase followingexposure of cells expressing either recombinant or endogenous KIAA0175to γ-irradiation or hydroxyurea.

Recombinant HA-KIAA0175 protein levels in Cos 7 cells were detectedeither without treatment or after treatment with either γ-irradiation orhydroxyurea. Lysates were loaded directly onto SDS polyacrylamide gelswithout prior immunoprecipitation of the HA fusion proteins. TheHA-fusion proteins were detected by probing with anti-HA antibodies.

Similarly, endogenous KIAA0175 protein levels in cells were detectedeither without treatment or after treatment with either γ-irradiation(HeLa cells) or hydroxyurea (Arent cells). Lysates were loaded directlyonto SDS polyacrylamide gels without prior immunoprecipitation of theKIAA0175 proteins. The KIAA0175 proteins were detected by probing withanti-KIAA0175 antibodies.

In total, the data revealed that treatment of Cos 7 cells withγ-irradiation or hydroxyurea caused an increase in KIAA0175 proteinlevels whether the KIAA0175 protein was expressed as a recombinantHA-fusion protein or was endogenously expressed.

Example 4 Kinetics of KIAA0175 Transcript Following Administration of anAnti-Sense Oligonucleotide

This example discloses that KIAA0175 mRNA levels may be downregulated byadministration of a KIAA0175 anti-sense oligonucleotide.

The following anti-sense oligonucleotides were used in the experimentsdisclosed herein: KIA175-545: (SEQ ID NO:1)5′-GAGGTCCCTGTGAGCATAGCCCTGG-3′ RC545: (SEQ ID NO:2)5′-GGTCCCGATACGAGTGTCCCTGGAG-3′ KIA175-736: (SEQ ID NO:3)5′-TGCCCATGCTCCAAACATCTGCCTC-3′ RC736: (SEQ ID NO:4)5′-CTCCGTCTACAAACCTCGTACCCGT-3′ KIA175-1182: (SEQ ID NO:5)5′-GGGTAGCACTGGCTTGTCCACAGGA-3′ RC1182: (SEQ ID NO:6)5′-AGGACACCTGTTCGGTCACGATGGG-3′ FITC: (SEQ ID NO: 11)5′-TCTGCTGCTGTCGACAACGAGTGTC-3′ AKT1-1548: (SEQ ID NO:12)5′-CCATAGTGAGGTTGCATCTGGTGCC-3′

HT1080 cells express endogenous KIAA0175. These cells were transfectedwith either the FITC (negative control) or AKT1 (positive control)oligonucleotides), 1182, or 545 oligonucleotides. Briefly, 100 nM of therespective anti-sense oligonucleotide was mixed with lipitoid 1transfection reagent (Huang et al., Chemistry and Biology 5(6):346-354(1998)) in a 1:3 volume to volume ratio in OPTI-MEM (GibcoBRL). Themixture was added to the HT1080 cells in complete medium (EMEM, 10% heatinactivated fetal bovine serum (FBS), 2 mM L-Glutamine, 100 U/mlPenicillin, and 1000 μg/ml Streptomycin) and the cells were incubated at37° C. for ^(˜)4-5 hours in the presence of the anti-senseoligonucleotides before changing to fresh complete medium. Total RNA wasextracted at day 1 or 24, 48, or 72 hours following transfection andmRNA levels of KIAA0175 were detected by Northern analysis. 20 μg oftotal RNA were subjected to electrophoresis on a denaturing agarose gel.RNA was transferred to nitrocellulose and KIAA0175 mRNA was detected byprobing with a ³²P-labeled, 0.7 kb KIAA0175 cDNA fragment. To controlfor equal loading of individual lanes, the blot was stripped of theKIAA0175 probe and re-probed with a ³²P-labeled G3PDH cDNA probeobtained from Clontech.

The data showed that any of the KIAA0175 anti-sense oligonucleotidestested, i.e., 1182, 545 or 736, were effective in reducing the level ofKIAA0175 mRNA expression. In contrast, reverse control primers (RC1182and RC736) as well as the control primers AKT and FITC were allineffective in reducing KIAA0175 mRNA expression. KIAA0175 mRNAexpression increased gradually over the time range of 24 to 72 hoursafter 545 and 1182 anti-sense oligonucleotide administration. FITC hadno effect on KIAA0175 mRNA expression over the time range tested.

Example 5 Western Analysis of KIAA0175, P53, ERK2 AND P21 Protein LevelsFollowing Administration of an Anti-Sense Oligonucleotide

This example discloses the effect of KIAA0175 anti-sense oligonucleotideadministration on KIAA0175, p53 and p21 protein levels.

In separate experiments, either HT1080 or HCT116 cells expressingendogenous KIAA0175 and p53 were transfected with either FITC, KIAA0175anti-sense oligonucleotides (i.e. 545 or 1182), or the correspondingreverse control oligonucleotides (RC or RC545). Total cell lysates werefractionated by SDS-polyacrylamide gel electrophoresis and transferredto nitrocellulose membranes. Specific proteins were detected by Westernanalysis using antibodies to either KIAA0175 (rabbit polyclonalantisera), p53, Erk2, or p21 antibodies as indicated. Antibodies againstp53, p21 and Erk2 were obtained from Santa Cruz Biotechnology, Inc.(Santa Cruz, Calif.).

In HT1080 cells, the administration of either of the KIAA0175 anti-senseoligonucleotides caused a reduction in both p53 and p21 protein levelsbut did not affect Erk2 protein level. Maximal 1182 anti-sense activityin HT1080 cells was observed at 48 hrs for both KIAA0175 and p53proteins. Similar results were obtained with HCT116 cells, disclosedherein, further suggesting the general nature of the effect of KIAA0175anti-sense across a variety of cell lines.

Example 6 Western Analysis of P53, ERK2 AND P21 Protein Levels Followingγ-Irradiation of Cells Transfected with a KIAA0175 Anti-SenseOligonucleotide

This example discloses the effect of a KIAA0175 anti-senseoligonucleotide on γ-irradiation-induced up-regulation of p53 and p21protein levels.

HT1080 cells expressing endogenous KIAA0175 were transfected with theKIAA0175 anti-sense oligonucleotide (1182) and the corresponding reversecontrol oligonucleotide (RC1182). After culture for 24-hours, thetransfected cells were treated with 10 Gy of γ-irradiation. At varioustimes after irradiation, cells were harvested and lysates prepared andsubjected to SDS-PAGE. p53, Erk2, and p21 proteins were detected byWestern analysis.

The data showed that administration of anti-sense oligonucleotide 1182blocked the γ-irradiation induced increase in p53 protein level observedwith the RC1182 oligonucleotide. Similarly, the 1182 oligonucleotidecaused a reduction of p21 protein levels. As a consequence of decreasedp53 expression levels, cells transfected with, e.g., the 1182 anti-senseoligonucleotide exhibit an increase in radiation sensitivity and, thus,the 1182 anti-sense oligonucleotide functions as a radiation sensitizer.

Example 7 Partial Loss of Cell Cycle Arrest Upon γ-Irradiation inKIAA0175 Anti-Sense Molecule Transfected HT1080 Cells

This Example discloses the effect of KIAA0175 anti-sense molecules oncell-cycle arrest in response to γ-irradiation.

Because both p53 and p21 are involved in the G1 to S phase cell cycletransition, the following experiments were performed in order to assessthe effect of KIAA0175 anti-sense molecules on cell cycle regulation.

Table 1 discloses the percentage of HT1080 cells in each stage of thecell cycle at various times following exposure to γ-irradiation. By 24hours, cells were arrested in both the G1 and G2 phases as evidenced bythe elevated percentage of cells in G0/G1 and G2/M phases as compared tothe diminished percentage of cells in the S phase. TABLE 1 0 24 50 72 hrafter γ-IR (8Gy) G0-G1 50.5 59.7 65.2 67.6 S 35.6 13.6 9.5 10.5 G2-M13.9 26.7 25.3 21.9

Table 2 discloses the effect of KIAA0175 anti-sense molecules onγ-irradiation induced cell-cycle arrest. In cells transfected withanti-sense molecules, there was a reduction in the accumulation of cellsin the G1 phase and a minimized reduction in the number of cells in theS phase as compared to cells transfected with a “reverse-control”anti-sense molecule as control. In contrast, the percentage of cellspresent in the G2/M phase transition was unaffected by theadministration of a KIAA0175 anti-sense molecule thus demonstrating thatthe effect of KIAA0175 anti-sense molecules in relieving theγ-irradiation induced cell-cycle arrest is specific for the G1 phase inHT1080 cells. TABLE 2 RC1182 1182 − + − + G0-G1 47.6 52.5 47.4 39.3 S 3314.9 25.7 18.6 G2-M 19.4 32.6 26.8 42.17

Without being limited to a specific theory of the present invention,these data are supportive of the essential role played by p53 inDNA-damage cell-cycle arrest at the G1 checkpoint and suggest that theadministration of KIAA0175 anti-sense molecules causes a decrease in p53gene expression and activity thereby alleviating the γ-irradiationinduced block in the G1-S cell-cycle transition.

Example 8 Sensitization of HCT-116 Cells to γ-Irradiation andHydroxyurea by Administration of KIAA0175 Anti-Sense Molecules

This Example discloses that KIAA0175 anti-sense molecules are effectivein sensitizing cells to both γ-irradiation and hydroxyurea.γ-irradiation treatment was performed as follows: On day one, HCT116cells were transfected with either the KIAA0175 anti-sense molecule No.545 (545) or with the 545 reverse control molecule (RC) and plated ontotissue-culture plates. On day three, one plate each of 545 and RCtransfected cells were treated with γ-irradiation (2, 4 or 8 Gy) andcultured an additional 2 hours. The γ-irradiation treated cells as wellas untreated control HCT116 cells were trypsinized, counted and seededin triplicate onto 6-well tissue culture plates to a density of 1000cells per well. After 11 days of culture, the cells were assayed forviability by staining with Crystal Violet. Total numbers of cellcolonies were then counted. Representative data showed thatadministration of the 545 anti-sense molecule increased the sensitivityof HCT116 cells to γ-irradiation, as evidenced by a decrease in thenumber of viable colonies arising from 545-treated cells as compared toRC-treated cells.

Hydroxyurea treatment was performed as follows: On day one, HCT116 cellswere transfected with either the KIAA0175 anti-sense molecule No. 545(545) or with the 545 reverse control molecule (RC) and plated ontotissue-culture plates. The following day (day two), one plate each of545 and RC transfected cells were treated with hydroxyurea (1 mM finalconcentration) and cultured an additional 24 hours. On day three, thehydroxyurea treated cells as well as untreated control HCT116 cells weretrypsinized, counted and seeded in triplicate onto 6-well tissue cultureplates to a density of 1000 cells per well. After 11 days of culture,the cells were assayed for viability by staining with Crystal Violet.Total numbers of cell colonies were then counted. Representative datashowed that administration of the 545 anti-sense molecule increased thesensitivity of HCT116 cells to hydroxyurea, as evidenced by a decreasein the number of viable colonies arising from 545-treated cells ascompared to RC-treated cells.

Example 9 Sensitization of Tumor Cells to Chemotherapeutic Drugs byAdministration of KIAA0175 Anti-Sense Oligonucleotides

This example discloses the sensitization of tumor cells byadministration of KIAA0175 anti-sense molecules.

The effect of KIAA0175 anti-sense molecules on cell sensitivity tochemotherapeutic drugs was tested on several cell lines using thelactate dehydrogenase (LDH) cytotoxicity assay essentially as follows:

Day 1: Cells were seeded in 4 separate 96 well plates, typically 5000cells/well and incubated at 37° C. and 5% CO₂.

Day 2: Cells were transfected with the KIA175-1182 and KIA175-545anti-sense oligonucleotides (SEQ ID NOs:5 and 1, respectively) as wellas the reverse complement controls, RC1182 and RC545 (SEQ ID NOs:6 and2, respectively) essentially as described in Example 4, with and withoutdrug. One plate (day 0) was left untransfected as a seeding control.

The transfection was carried out using a lipid vehicle for delivery asdescribed in WO 01/16306, hereby incorporated in its entirety. Briefly,the transfection used agents known as “lipitoids” and “cholesteroids”,described, for example, in PCT publications WO 01/16306, WO 98/06437 andWO 99/08711, based on U.S. Ser. Nos. 60/023,867, 60/054,743, and09/132,808, which are also hereby incorporated by reference. Theselipid-cationic peptoid conjugates are shown in these references to beeffective reagents for the delivery of plasmid DNA to cells in vitro.Any of the carriers described in the above-referenced applications aresuitable for use in transfection of the oligonucleotides describedherein.

These compounds may be prepared by conventional solution or solid-phasesynthesis. In one such procedure, as described in WO 99/08711, citedabove, the N-terminus of a resin-bound peptoid is acylated with a spacersuch as Fmocaminohexanoic acid or Fmoc-3-alanine. After removal of theFmoc group, the primary amino group is reacted with cholesterolchloroformate to form a carbamate linkage. The product is then cleavedfrom the resin with trifluoroacetic acid and purified by reverse-phaseHPLC. A fatty acid-derived lipid moiety, such as a phospholipid, may beused in place of the steroid moiety. The steroid or other lipid moietymay also be linked to the peptoid moiety by other linkages, of anyeffective length, readily available to the skilled practitioner.

Depending on the cell type, different lipid vehicles were used fordifferent lengths of time for transfection. However, the transfectiontime did not exceed 24 hrs. The transfection was carried out in completemedium and the final anti-sense oligonucleotide concentration was 300 nMper well. In the wells with drug, the drug was added to the culture atthe beginning of the transfection.

Starting on day 3: cells were recovered, 1 plate/day and release of LDHinto the supernatant was measured using a kit from Roche according tomanufacturer's instructions (Roche Diagnostics, Basel, Switzerland)(data labeled as day 1, 2, 3).

For each sample, the data are expressed as a ratio of the level of LDHin wells containing cells transfected with anti-sense (AS) over thelevel of LDH in wells containing cells transfected with the reversecomplement controls (RC) (ie:AS/RC). Thus, cells that become sensitizedto drug will die and release LDH into the supernatant. An increase inthe ratio signifies increased cell death and thus, higher levels of LDHin the media. A ratio higher than 1.5 signifies an effect of theanti-sense over the reverse complement control.

The data summarized in Table 3 demonstrates that neither KIA175-1182 norKIA175-545 has an effect on the 184B5 normal breast epithelial cellline. By contrast, Table 4 shows that KIA175-545 sensitizes theMDA-MB-231 metastatic breast cancer cells to the effects of Cisplatinand to Campthesin (CPT). It did not appear to sensitize cells to theeffects of Doxorubicin (Doxo). The data in Table 4 also shows that bothanti-sense molecules alone caused an increase in cell death in thisbreast cancer cell line, suggesting that KIAA0175 may be targeteddirectly by inhibitors and such an inhibitor may be used therapeuticallyin the absence of other chemotherapeutic drugs. TABLE 3 Effect ofKIAA0175 Antisense −/+ Cisplatin on Normal Breast Epithelial Cell Line184B5 Bcl2 AS KIA175-545 + Day (+control) KIA175-1182 KIA175-545 7 μMCisplatin 0 0.0000 0.0000 0.0000 0.0000 1 1.2909 1.0223 1.1152 1.0691 21.5415 0.9065 1.1258 1.1928 3 1.3150 1.0663 1.2934 1.1284

TABLE 4 Effect of KIAA0175 Antisense −/+ Drug on Breast Cancer Cell LineMDA-MB-231 KIA175-545 + KIA175-545 + KIA175-545 + Day Bcl2 ASKIA175-1182 KIA175-545 Cisplatin Doxo CPT 0 1.03 0.76 0.84 0.71 0.890.79 1 1.04 2.04 1.05 1.10 1.05 1.12 2 2.2  1.67 1.37 1.72 1.39 1.69 32.23 1.52 1.62 1.95 1.34 1.88Cis: Cisplatin;Doxo: Doxorubicin;CPT: Campthesin.

Example 10 Phosphorylation of KIAA0175 by ATM in Response to GammaIrradiation

Ataxia telangiectasia mutated (ATM) is one of a subfamily of PI-3-likekinases and plays a central role in the DNA damage response. ATM isthought to primarily control the response to γ irradiation. The aminoacid motifs SQ or TQ are the preferred sites of phosphorylation of ATMin many of its reported substrates, including p53, Chk2, Mdm2, etc.(Kim, S. T. et al., J. Biol. Chem. 1999 274(53):37538-43). KIAA0175contains 3 SQ (Ser 100, Ser 125, Ser 391) sites.

GST-fusion proteins containing amino acids 42-182, 227-325, and 327-517of KIAA0175 were expressed and purified from Escherichia coli and testedfor their ability to act as substrate for ATM in vitro. Proteins werephosphorylated with ATM immunoprecipitates. A fusion including aminoacids 327-517 harboring S³⁹¹Q was a substrate for ATM. In contrast,phosphorylation of two other KIAA0175 fusions, amino acid 42-182(containing S¹⁰⁰Q and S¹²⁵Q) and amino acids 227-325 with no SQ sites,were not phosphorylated. In addition, KIAA0175 (327-517) phosphorylationwas comparable to that seen with several known substrates of ATMincluded in the same assay, GST-p53 (1-106), GST-Chk2(1-222), andGST-hDM2 (330-491).

To further test whether KIAA0175 phosphorylation by ATM is regulated byDNA damage and to further map the putative site of phosphorylation,serine 391 was mutated to alanine. ATM immunoprecipitated from cellstreated with γ-irradiation (1R) showed increased phosphorylation towardsKIAA0175 (327-517) by ˜2-3 fold, similar to that observed with p53 orChk2. Mutation of serine 391 to alanine completely abolished theirradiation induced phosphorylation by ATM. These results demonstratedthat ATM can directly phosphorylate KIAA0175 in vitro in response to DNAdamage and confirmed that the primary site of phosphorylation mapped toserine 391.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such specific embodimentsand equivalents are intended to be encompassed by the following claims.

All patents, published patent applications, and publications citedherein are incorporated by reference as if set forth fully herein.

1-26. (canceled)
 27. A method of treating neoplastic disease in a mammalin need of said treatment, comprising administering to said mammal aKIAA0175 inhibitor is selected from the group consisting of ananti-sense molecule, a ribozyme, an antibody, an antibody fragment, aprotein, a polypeptide and a small molecule such that said neoplasticdisease is reduced in severity.
 28. The method of claim 27 wherein saidKIAA0175 inhibitor is an anti-sense molecule.
 29. The method of claim 27wherein said anti-sense molecule or the complement thereof comprises atleast 17 consecutive nucleic acids of the sequence of SEQ ID NO:9. 30.The method of claim 27 wherein said anti-sense molecule or thecomplement thereof hybridizes under high stringency conditions to thesequence of SEQ ID NO:9.
 31. The method of claim 27 wherein saidanti-sense molecule comprises a nucleic acid sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5.
 32. Themethod of claim 27, wherein said treating comprises increasing thechemosensitivity and/or radiosensitivity of a mammalian cell, whereinsaid mammalian cell is a tumorigenic cell.
 33. The method of claim 32wherein said increase in chemosensitivity and/or radiosensitivity isdetermined by a measurement selected from the group consisting ofmeasuring a reduction in γ-irradiation or hydroxyurea induced p53 or p21protein levels, measuring a reduction in γ-irradiation or hydroxyureainduced cell cycle arrest and measuring an increase in γ-irradiation orhydroxyurea induced cell sensitization.